A型塞内卡病毒衣壳蛋白的原核表达及其五聚体的组装

Prokaryotic expression of capsid protein of Seneca virus type A and assembly of its pentamer

为实现A型塞内卡病毒(SVA)衣壳蛋白在大肠杆菌中的可溶性表达并组装为五聚体,本试验以带His标签和SUMO(小泛素样修饰系统)标签的pSMK及pSMA为载体,将塞内卡病毒衣壳蛋白基因VP0、VP1和VP3构建为重组质粒pSMA-VP0、pSMK-VP1、pSMK-VP3,在大肠杆菌原核表达系统中对其进行共表达并优化诱导表达条件,包括诱导温度、诱导剂IPTG浓度、诱导前细菌D600值以及诱导时间,纯化后获得SVA衣壳蛋白后进行免疫印迹分析,之后利用His-SUMO蛋白酶切除标签蛋白,在体外进行组装和鉴定。结果,A型塞内卡病毒衣壳蛋白VP0、VP1、VP3在20℃下、D600值为1.1、 IPTG浓度为0.4 mmol/L、诱导12 h后蛋白表达效果最好。经超声破碎、镍柱纯化后获得VP0、VP1、VP3蛋白,免疫印迹结果表明,目的蛋白能与SVA阳性血清发生特异性反应,证实该蛋白具有良好的反应原性。动态光散射和扫描电镜检测结果显示,去除His-MUMO后还原天然构象的目的蛋白可组装成五聚体。上述研究结果为制备SVA病毒样颗粒奠定了物质基础。

In order to achieve the soluble protein of Seneca virus type A capsid protein from E.coli and initially assembled as pentamers,the SVA capsid protein gene was constructed into p SMK and pSMA plasmid with His-tag and SUMO(small ubiquitin-modified system) tags,and finally we obtained pSMA-VP0,p SMK-VP1,and p SMK-VP3.The plasmids were co-transformed into E.coli competent cells and induced co-expression of capsid protein.The expression conditions was optimized,including induction temperature and concentration of IPTG,D600 value and induction time,immunoblot analysis after obtaining SVA capsid protein,and then the tagged protein was excised by His-SUMO protease and assembled and identified in vitro under certain circumstances.In result,the SVA capsid proteins VP0,VP1 and VP3 were expressed with highest level when D600 value up to 1.1,IPTG concentration is 0.4 mmol/L,at 20 ℃and incubation time is 12 h.VP0,VP1 and VP3 proteins were purified by nickel column after ultrasonication of cell lysate.The results of immunoblotting showed that the target protein could specifically recognized by anti-SVA serum,which confirmed that the protein had good reactivity,The results of dynamic light scattering and transmission electron microscope showed that the removal of fusion tag from capsid protein can form pentamer.This study result laid the material foundation for SVA virus-like particles.